Expression vector suitable for expression of a coding sequence for gene therapy

ABSTRACT

Provided is an expression vector for gene therapy having a novel combination of transcriptional regulatory elements, including a promoter, an enhancer, an intron, an untranslated region (UTR) and a locus control region (LCR). The expression vector enables sustained expression of a liver tissue-specific gene, and thus, can be effectively used for treating thrombosis, hemophilia, liver cancer, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. patent applicationSer. No. 12/867,716 filed Aug. 13, 2010, which is a National Stage ofInternational Application No. PCT/KR2008/000870 filed Feb. 14, 2008, thecontents of all of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to an expression vector for gene therapy,and more particularly, to an expression vector for gene therapyincluding a novel combination of transcriptional regulatory elements(cis-regulatory elements or cis-acting elements) which include apromoter, an enhancer, an intron, an untranslated region (UTR) and alocus control region (LCR), said vector being capable of sustaining theexpression of a target gene at a high level in the liver.

BACKGROUND OF THE INVENTION

The functions of the liver include blood storage, sugar conversion andthe secretion of various cytokines, as well as the expression of genesthat affect many diseases such as genetic, cardiovascular, metabolic,hematologic and cancerous disorders. Various liver-specific diseases,such as infectious hepatitis, have been studied as targets for genetherapy.

A liver cell maintains a long lifespan after its formation, hasreceptors of most viral gene transporters, and is directly connected tothe bloodstream, enabling an easy approach of a drug, and thus, theliver is recognized as an important candidate organ for gene therapy.

Hemophilia is a degenerative hemorrhagic disease caused by thedeficiency of factor VIII (FVIII, f8) or factor IX (FIX, f9) genelocated on the X chromosome, and is classified into Hemophilia A (FVIIIdeficiency) or B (FIX deficiency) depending on the mutated or deletedgene. As for drugs for treating Hemophilia A or B, the therapeuticeffect can be expected only when FVIII or FIX is continuously expressedat a level of 1-5% or more of the normal blood concentration thereof(100-200 ng/ml and 500 ng/ml, respectively).

In recent gene therapy studies of hemophilia B, it has been reportedthat the introduction of adeno-associated virus (AAV) carrying human FIX(hFIX) gene into a hemophilia B mouse model led to the expression ofhFIX protein at a level of up to 1,500-1,800 ng/ml (Snyder, R. O., Nat.Med., 5: 64-70 (1999); Manno, C. S., Nat. Med., 12: 342-347 (2006)),while hFIX protein was expressed at the level of up to 730 ng/ml in ahemophilia B dog model (Arruda, V. R., Blood, 105: 3458-3464 (2005)).However, when such highly efficient expression vector for an animalmodel was clinically applied to a human patient, the blood concentrationof FIX was only 185 ng/ml or less, which is less than 500 ng/ml, thethreshold value for an effective clinical treatment, and besides, theproblem was that the hFIX expression level in a human subject was notsustained but transient (Kay, M. A., Nat. Genet., 24: 257-261 (2000);Manno, C. S., Nat. Med., 12: 342-347 (2006)). A trend similar to theabove has been found in the treatment models for hemophilia A.

The results of clinical tests for the treatment of a genetic diseasesuch as hemophilia show that it is prerequisite to develop an efficienttissue-specific expression vector capable of keeping a high andsustained level of expression of a therapeutic gene in a specific tissuesuch as a liver tissue. Further, the escaping from humoral and cellularimmune response against a vector and the induction of immune toleranceto an expressed protein have been recognized as critical factors for thesuccessful gene therapy. For example, in order to raise the expressionlevel of normal FVIII or FIX to a threshold value effective forsuccessful clinical treatment, the injection of a high dose of viruscarrying FVIII or FIX gene as well as the suppression of in vivo immuneresponse also have to be considered. For such approaches to success,however, it is prerequisite to enhance the gene expression efficiency bythe improvement of an expression vector.

Lipoprotein (a) produced only in the liver is another important targetfor the development of a liver tissue-specific expression vector.Lipoprotein (a) is formed through the binding of apolipoprotein (a), aglycoprotein, with apo B-100, a major protein component of low-densitylipoprotein (LDL) (Fless, G. M., J. Biol. Chem., 261: 8712-8717 (1986)).Apolipoprotein (a) is responsible for cholesterol transportation invivo, and the increase of the lipoprotein (a) concentration in theplasma has been reported to be a major risk factor of arteriosclerosisand cardiac diseases (Armstrong, V. W. et al, Arteriosclerosis, 62:249-257 (1986); Assmann, G., Am. J. Cardiol., 77: 1179-1184 (1996)).Apolipoprotein (a) contains two types of kringle domains similar toplasminogen kringles IV and V, together with an inactive protease-likedomain. It is well known that proteins having a kringle structure mayinhibit tumor neovascularization and metastasis (Folkman J., N Eng JMed, 285: 1182-1186 (1971); Falkman J, Klagsbrun M., Science, 235:442-447 (1987); Scapaticci F A., J Clin Oncol., 20: 3906-3927 (2002)).Recently, the present inventors as well as other researchers have foundthat the kringle domains of apolipoprotein (a) have anti-cancer andanti-metastasis activities owing to their significant anti-angiogenesisactivity (Sculter V et al, Arterioscler Thromb Vasc Biol, 21: 433-438(2001); Trieu U N and Uckun F M., Biochem Biophys Res Commun., 257:714-718 (1999); Kim J S et al, J. Biol Chem., 278: 29000-29008 (2003);Yu H K et al, Cancer Res., 64: 7092-7098 (2004); Kim J S et al, BiochemBiophys Res Commun., 313: 534-540 (2004); Lee K et al, Hepatology, 43:1063-1073 (2006)).

In anti-metastasis and anti-cancer therapy, it has been widelyrecognized that a mode of therapy which selectively acts on an affectedsite would be most effective. Therefore, it is very important to developa vector having the ability of tissue-specific and continuous geneexpression for anticancer therapy, e.g., for effective gene therapy forliver cancer or metastatic liver tumors.

As described above, tissue-specific and sustained gene expression is thekey for efficient gene therapy, which requires the development of anovel, improved expression vector. This may be achieved by theimprovement of the transcriptional regulatory elements (cis-regulatoryelements or cis-acting elements) of such an expression vector.

Examples of common transcriptional regulatory elements include apromoter, an enhancer, an intron, an untranslated region, a locuscontrol region, and others.

Used for such an expression vector for liver tissue-specific expressionare promoters of phosphoenolpyruvate carboxykinase (PEPCK), agluconeogenesis enzyme (Yang, Y. W., J. et al, Gene Med., 5(5): 417-424(2003)), α1-antitrypsin protease, albumin, FVII, organicanion-transporting polypeptide-C(OATP-C), hepatitis B virus core(Kramer, M. G., et al, Mol. Ther., 7(3): 375-385 (2003)), andthyroxin-binding globulin (Wang, L., et al, Proc. Natl. Acad. Sci., 96:3906-3910 (1999)); and enhancers of albumin (Kang, Y., et al, Blood,106(5): 1552-1558 (2005)), phenylalanine hydroxylase (PAH) andα1-microglobulin/bikunin precursor (AMBP) (Wang, L., et al., Mol. Ther.,1(2): 154-158 (2000)).

The FVII promoter having a size of about 500 bp is transcriptionallyactivated in the liver at a level 10-fold or more higher than in othertissues, due to the binding of liver-enriched HNF-4 (hepatocyte nuclearfactor-4). It has been reported that most transcription factors aremostly bound to a 300-bp fragment of the 3′-end of the FVII promoter(Greenberg, D., et al, Proc. Natl. Acad. Sci., 92: 12347-12351 (1995)).When a 315-bp fragment of the 5′-end of a FVII promoter with a size of501 bp is truncated, the liver-specific activity of the promoterincreases by about 30%, but it decreases by about 20-30% when a 210-bpfragment is truncated (Pollak, W. S., et al, J. Biol. Chem., 271(3):1738-1747 (1996)).

An organic anion-transporting polypeptide-C (OATP-C) promoter having asize of about 900 bp is transcriptionally activated in the liver at alevel 3-fold or more higher than in other tissues, due to liver-enrichedHNF-1α binding. It has been reported that most transcription factors arebound to a 440-bp fragment of the 3′-end of the promoter (Jung, D., etal, J. Biol. Chem., 276: 37206-37214 (2001)).

The activity of a promoter can be raised by the action of an enhancer.Phenylalanine hydroxylase (PAH) enhancer, which has a size of about 230bp and HNF-1 binding sites, is located −3.5 kb upstream of the 5′-end ofthe PAH gene. It has been reported that the PAH enhancer increases theactivity of the promoter bound thereto by 4-fold or more, due to thepresence of liver-enriched HNF-1 binding sites (Lei, X. D., et al, Proc.Natl. Acad. Sci., 95: 1500-1504 (1998)).

AMBP (α1-microglobulin/bikunin precursor) enhancer has a size of about400 bp, which extends from −2945 to −2539 bp upstream of the 5′-end ofthe AMBP gene. It is mainly composed of HNF-1, 2, 3 and 4 binding sites,and its major active region corresponds to the −2802 to −2659 bp segmentthereof (Route, P., et al, Biochem. J., 334: 577-584 (1998)). Generally,it has been reported that the AMBP enhancer increases the promoteractivity by about two or three times.

Untranslated regions (UTRs) located at the 5′- and 3′-ends of a gene areresponsible for the structural stabilization of gene mRNA (Holcik, M.,Liebhaber S. A., Proc Natl Acad Sci USA., 94: 2410-2414 (1997);Chkheidze, A. N., et al, Mol Cell Biol., 19: 4572-4581 (1999)). It hasbeen reported that the polyadenylation signal sequence in 3′ UTR alsosignificantly contributes to the structural stabilization of the mRNA(Kolev, N. G., et al, Genes Dev., 19: 2583-2592 (2005)).

A eukaryotic gene is composed of exons which are translated to proteins,and introns which are untranslated sequences between the exons. An mRNAprecursor primarily transcribed from DNA is converted to a mature mRNAby the removal of such introns through splicing. Such introns include asplicing donor starting with GT(U) and a splicing acceptor ending withAG. Further, present in the 3′-end of an intron are, among others, apolypyrimidine tract, a splicing factor, an snRNP-binding branchsequence, a triple guanine repeat sequence (G-triple motif), which playcritical roles in forming a spliceosome which is a complex of RNA andintron splicing enzyme (Pagani, F., et al, Nat. Rev. Genet., 5: 389-396(2004)).

An intron can be applied as a transcriptional regulatory element forsustained, efficient gene expression, when combined with a promoter andan enhancer. The intron is involved in enhancing the gene expressionefficiency and transcription efficiency by its binding withtranscription factors (Liu, K., et al, Proc. Natl. Acad. Sci., 92:7724-7728 (1995); LeBlanc, S. E., et al, J. Biol. Chem., (2005)), andalso in the enhancement of the post-transcriptional protein translationefficiency (Moore, M. J., Cell, 108: 431-434 (2002)). It has beenreported that intron 1 of hFIX causes an increase in the expression ofhFIX protein (Kurachi, S., et al, J. Biol. Chem., 270: 5276-5281(1995)).

Anti-coagulation proteins such as antithrombin and plasminogen, as wellas coagulation factors such as prothrombin are expressed in the liver.On analyzing the introns of the gene of a thrombosis or hemophiliapatient deficient of such proteins, various missense and/or nonsensemutations have been observed, which suggests that the introns of theseproteins play critical roles in the gene expression in the liver(Jochmans, K., et al, Blood, 84: 3742-3748 (1994)).

Locus control regions (LCRs) have been found to be present in at least36 types of mammals including human, rat, rabbit, goat and the like.LCRs are nucleotide sequences having a DNase I-sensitive region which istissue-specific for transcription factors. The functions of human betaglobin LCRs are well known (Harju S. et al, Exp. Biol. Med. 227: 683-700(2002)).

A hepatocyte control region (HCR) is known for its ability to enhancethe liver-specific gene expression, and found downstream ofapolipoprotein E (ApoE) gene. HCR has a DNase I-sensitive region whichbinds with liver-specific transcription factors. The HCR acts as an LCRfor liver-specific expression of ApoE gene. The ApoE HCR has sites thatare activated when it binds with various factors such as HNF3α, HNF4,GATA-1, C/EBP, TF-LF2 and Alu-family. Among these sites, the HNF3αbinding site showing hypersensitivity to DNase I has a TGTTTGC motif,and the link between the first G and the second T is cleaved by DNase I(Dang Q., et al, J. Biol. Chem. 270: 22577-22585 (1995)). Actually, ithas been reported that the introduction of ApoE HCR into an expressionvector leads to enhanced expression of hFIX in an animal model (Miao C.H., et al., Mol. Ther. 1: 522-532 (2000)).

Proteins having sequences similar to HCR of human ApoE gene includingthe TGTTTGC motif, apolipoprotein A-I, apolipoprotein B, transferin,α-fetoprotein, α1-antitrypsin and the like in human, and α-fetoprotein,albumin and the like in mouse have been reported (Dang Q., et al., J.Biol. Chem. 270: 22577-22585 (1995)).

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide apolynucleotide which can be used as a transcriptional regulatory elementfor sustained expression of a liver tissue-specific gene at a highlevel.

It is another object of the present invention to provide an expressionvector for sustained expression of a liver tissue-specific gene at ahigh level.

In accordance with an aspect of the present invention, there is provideda polynucleotide having the nucleotide sequence of SEQ ID NO: 42.

In accordance with another aspect of the present invention, there isprovided a polynucleotide including a polynucleotide having thenucleotide sequence of SEQ ID NO: 42 and at least one polynucleotideselected from the group consisting of polynucleotides having thenucleotide sequences of SEQ ID NOS: 44 and 45 operably linked to thepolynucleotide of SEQ ID NO: 42.

In accordance with another aspect of the present invention, there isprovided a polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to 57.

In accordance with another aspect of the present invention, there isprovided a polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to 61.

In accordance with another aspect of the present invention, there isprovided an expression vector including a transcriptional regulatoryelement and a coding sequence operably linked to and under control ofthe transcriptional regulatory element, wherein the transcriptionalregulatory element includes:

1) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42;

2) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42 andat least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 44 and 45operably linked to the polynucleotide of SEQ ID NO: 42;

3) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42 andat least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to 57operably linked to the polynucleotide of SEQ ID NO: 42;

4) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42 andat least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to 61operably linked to the polynucleotide of SEQ ID NO: 42;

5) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42; atleast one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 44 and45; and at least one polynucleotide selected from the group consistingof polynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to57, said polynucleotides being operably linked to each other;

6) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42; atleast one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 44 and45; and at least one polynucleotide selected from the group consistingof polynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to61, said polynucleotides being operably linked to each other;

7) a polynucleotide having the nucleotide of SEQ ID NO: 42; at least onepolynucleotide selected from the group consisting of polynucleotideshaving the nucleotide sequences of SEQ ID NOS: 46 to 57; and at leastone polynucleotide selected from the group consisting of polynucleotideshaving the nucleotide sequences of SEQ ID NOS: 58 to 61, saidpolynucleotides being operably linked to each other; or

8) a polynucleotide having the nucleotide of SEQ ID NO: 42; at least onepolynucleotide selected from the group consisting of polynucleotideshaving the nucleotide sequences of SEQ ID NOS: 44 and 45; at least onepolynucleotide selected from the group consisting of polynucleotideshaving the nucleotide sequences of SEQ ID NOS: 46 to 57; and at leastone polynucleotide selected from the group consisting of polynucleotideshaving the nucleotide sequences of SEQ ID NOS: 58 to 61, saidpolynucleotides being operably linked to each other.

In accordance with a further aspect of the present invention, there isprovided an expression vector including a transcriptional regulatoryelement and a coding sequence operably linked to and under control ofthe transcriptional regulatory element, the transcriptional regulatoryelement including a promoter and a polynucleotide which is operablylinked to the promoter and selected from the group consisting of: atleast one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to 57;at least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to 61;and at least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to 57operably linked to at least one polynucleotide selected from the groupconsisting of polynucleotides having the nucleotide sequences of SEQ IDNOS: 58 to 61.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention taken inconjunction with the following accompanying drawings, which respectivelyshow:

FIG. 1A: schematic diagrams of pCR-FVII Pro plasmid containing FVIIpromoter, pCR-OATP-C Pro plasmid containing organic anion-transportingpolypeptide-C (OATP-C) promoter, and pCR-AAT Enh/Pro plasmid containingα-antitrypsin (AAT) promoter/enhancer;

FIG. 1B: schematic diagrams of mRLuc luciferase expression vectorspmRL-FVII Pro, pmRL-FVIIΔ Pro and pmRL-OATP-C Pro containing FVIIpromoter, truncated FVII (FVIIΔ) promoter and OATP-C promoter,respectively;

FIG. 2A: a histogram showing the expression efficiencies of FVIIpromoter and OATP-C promoter in: a human umbilical vein endothelial cellline (HUVEC); a human hepatocellular carcinoma cell line (Huh-7); ahuman kidney cell line (HEK293); a human lung adenocarcinoma epithelialcell line (A549); and a human cervical carcinoma cell line (HeLa);

FIG. 2B: schematic diagrams of luciferase expression cassettescontaining FVII promoter and FVIIΔ promoter, and a histogram comparingthe expression efficiencies of the promoters;

FIG. 2C: a histogram showing the expression efficiencies of FVIIΔpromoter, OATP-C promoter, hepatitis B virus core protein (HBcP)promoter, endoglin (Endoglin) promoter, cell adhesion molecule (ICAM2)promoter and tyrosine kinase receptor (Tie2) promoter, in a mannercomparative with CMV promoter;

FIG. 3A: schematic diagrams of pCR-PAH enh and pCR-AMBP enh plasmidscontaining phenylalanine hydroxylase (PAH) enhancer andα1-microglobulin/bikunin precursor (AMBP) enhancer, respectively;

FIG. 3B: schematic diagrams of pmRL-PF, pmRL-AF, pmRL-PAF, pmRL-PO,pmRL-AO and pmRL-PAO expression vectors, which are constructed byinserting at least one selected from PAH enhancer and AMBP enhancer intopmRL-FVIIΔ Pro expression vector containing FVIIΔ promoter orpmRL-OATP-C Pro expression vector containing OATP-C promoter;

FIG. 4A: histograms showing the in vitro luciferase expression levels inA549, HeLa and Huh-7 depending on the variation in the mode ofcombination of PAH enhancer and AMBP enhancer with FVIIΔ, OATP-C, Tie-2and ICAM2 promoters;

FIG. 4B: a histogram showing the luciferase expression efficiencies andliver specificities in HEK293, A549, HeLa, Huh-7, human hepatoma cellline (Hep3B) and HUVEC when AMBP enhancer was combined with FVIIpromoter, in a manner comparative with a CMV promoter;

FIG. 4C: histograms showing the luciferase expression efficiencies inliver tissues for expression vectors carrying expression cassettescontaining PAH enhancer, AMBP enhancer, CMV enhancer and SV40 enhancertogether with the promoters shown in FIG. 4A after a mixture of eachexpression vector with polyethyleneimine (PEI) or in vivo jetPEI wasinjected into a mouse tail vein;

FIG. 5: schematic diagrams of hFIX expression plasmid including no UTR(FIX) and hFIX expression plasmid containing UTRs (FIXUTR)((a)), andFIXUTR plasmids containing various introns ((b), (c) and (d)) (SD,splicing donor; SA, splicing accepter; (G)₃, triple guanine motif; BS,branch sequence);

FIG. 6A: ELISA results showing the expression level of hFIX protein inthe plasma samples obtained from the mice injected through tail veinwith the expression vectors carrying CMV-hFIXUTR-Syn1int,CMV-hFIXUTR-Syn2int, CMV-hFIX and CMV-hFIXUTR expression cassettes;

FIG. 6B: ELISA results showing the expression level of hFIX protein inthe plasma samples obtained every week from immunodeficient mice inwhich 1×10⁹ infectious particles (IP) of recombinant adeno-associatedviruses carrying CMV-hFIXUTR-Syn1int expression cassettes are injected;

FIG. 7A: a schematic diagram showing a luciferase expression cassettecontaining an intron into RLuc luciferase gene controlled under TKpromoter;

FIG. 7B: a histogram showing luciferase expression levels in Hep3B,HEK293 and A549 cells transfected with expression vectors carrying theexpression cassettes containing various introns of FIG. 7A;

FIG. 8A: reverse-transcription PCR (RT-PCR) results showing mRNAexpression levels of RLuc and β-actin in total RNA extracted from A549cells transfected with the expression vectors of FIG. 7B;

FIG. 8B: a histogram showing mRNA expression levels of RLuc normalizedwith β-actin for the mRNA band intensities of RLuc and β-actin obtainedin FIG. 8A;

FIG. 9A: ELISA results showing the expression level of hFIX protein inthe plasma samples obtained from mice injected through tail vein withthe expression vectors carrying CMV-hFIXUTR-Syn1AT, CMV-hFIXUTR-Syn2AT,CMV-hFIXUTR-NA, CMV-hFIXUTR-ΔNAL, CMV-hFIXUTR-ΔNAS expression cassettes,and a CMV-hFIXUTR-0.3 kbFIXint (CMV-hFIXm2) expression cassettecontaining a 0.3 kb FIX intron as a control;

FIG. 9B: the results of Western blot analysis showing a hFIX proteinexpression level in HEK293T transfected with expression vectors carryingCMV-hFIX, CMV-hFIXUTR and CMV-hFIXUTR-ΔNAL expression cassettes;

FIG. 10: a schematic diagram showing distribution of motifs identical orsimilar to the TGTTTGC motif found in LCR of Apo E gene, at 5′-endflanking regions of α-fetoprotein, AAT and albumin gene;

FIG. 11A: schematic diagrams of pCR-HCR and pCR-HCRm plasmids containingHCR (known as a hepatocyte control region of Apo E gene) and HCRm (knownas a minimum structure HCR truncated with the TGTTTGC motif),respectively, and pCR-AAT7800lcr/pCR-AAT108lcr, pCR-AFP3800lcr andpCR-E6lcr plasmids containing the respective TGTTTGC motifs ofα-fetoprotein, AAT and albumin;

FIG. 11B: schematic diagrams of a pBS-HCRm-AATenh/pro (HmA) plasmidconstructed by inserting HCRm, AAT enhancer and AAT promoter into apBluescript II vector, and a pBS-HCR-AATpro (HA) plasmid constructed byinserting HCR and AAT promoter into a pBluescript II vector;

FIG. 12A: schematic diagrams showing expression cassettes obtained byintroducing various LCRs (HCR, HCRm, AFP3800, AAT7800, AAT108 and E6)into hFIX expression cassettes containing PAH enhancer, FVIIΔ promoterand intron Syn1PLA;

FIG. 12B: ELISA results showing the expression level of hFIX protein inthe plasma samples obtained from the mice injected through tail veinwith expression vectors carrying the expression cassettes of FIG. 12Aand a CMV-hFIXUTR-Syn1PLA expression vector;

FIG. 13A: schematic diagrams showing expression cassettes containing acombination of PAH enhancer and FVIIΔ promoter (PF) or a combination ofPAH enhancer, AMBP enhancer and FVIIΔ promoter (PAF), NAL intron, andAAT108 LCR; a HmA (HCRm-AAT enh/pro)-hFIXUTR-ΔNAL expression cassette;and a HA (HCR-AATpro)-hFIXUTR-1.4kbFIXint expression cassette;

FIG. 13B: ELISA results showing the expression level of hFIX protein inthe plasma samples obtained from the mice injected through tail veinwith expression vectors carrying the expression cassettes of FIG. 13A,and a CMV-hFIXUTR-ΔNAL expression vector;

FIGS. 13C and 13D: the clotting time and activity measured by anactivated partial thromboplastin time (APTT) method using the plasmasamples obtained in FIG. 13B;

FIGS. 14A and 14B: the results showing the expression level of hFIXprotein by ELISA and the clotting activity measured by APTT method,respectively, for plasma samples obtained from hemophilia B miceadministered through portal vein with 2×10⁹ IP of recombinantadeno-associated viruses carrying AAT108-PF-hFIXUTR-ΔNAL andAAT108-PAF-hFIXUTR-ΔNAL expression cassettes, and commonHA-hFIXUTR-1.4kbFIXint and CMV-hFIXUTR-1.4kbFIXint expression cassettesas a control;

FIG. 15A: schematic diagrams of expression vectors constructed byintroducing various LCRs (HCR, HCRm, AFP3800, AAT7800, AAT108 and E6)into PF-LK68-ΔNAL-UTR expression cassettes;

FIG. 15B: schematic diagrams of an AAT108-PF/PAF-LK68 expression vector,an AAT108-PF/PAF-LK68-UTR expression vector, and anAAT108-PF/PAF-LK68-ΔNAL-UTR expression vector;

FIG. 16A: ELISA results showing the expression level of LK68 protein inthe plasma samples obtained from the mice injected through tail veinwith expression vectors carrying PF-LK68-ΔNAL-UTR expression cassettesintroduced various LCRs (HCR, HCRm, AFP3800, AAT7800, AAT108 and E6);

FIG. 16B: ELISA results showing the expression level of LK68 protein inthe plasma samples obtained from the mice injected through tail veinwith expression vectors carrying AAT108-PF/PAF-LK68 expression cassettesintroduced UTR or UTR and intron ΔNAL;

FIG. 17A: Western blot analysis results for LK68 protein expressionlevels in HEK293 cells infected with replication-defective adenovirusescarrying CMV-LK68 and CMV-LK68-ΔNAL-UTR expression cassettes atdifferent multiplicity of infection ratios (MOI), and in theirs culturemedia; and

FIG. 17B: a histogram for band intensities obtained from the Westernblot analysis results of FIG. 17A to show an effect of UTR and intron ongene expression.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Further, all documents mentionedherein are incorporated by reference in their entireties.

The term “expression vector” as used herein, is intended to comprehendan aggregation (expression cassette or construct) including a codingsequence, a promoter, and optionally one or more transcriptionalregulatory elements operably linked to the coding sequence, or a vectorincluding the aggregation.

The term “coding sequence” as used herein means a DNA sequence encodingan amino acid or a functional RNA.

The term “transcriptional regulatory element (cis-regulatory element orcis-acting element)” as used herein means a nucleotide sequence locatedupstream, within, or downstream the coding sequence, which controls theRNA transcription, the processing, the stability and the subsequenttranslation of the transcribed RNA. The transcriptional regulatoryelement includes a promoter, an enhancer, an intron, 5′- and3′-untranslated regions (UTRs), and a locus control region (LCR).

The terms “promoter” and “enhancer” as used herein are DNA sequencesrepresenting fragments that are needed to transcript the coding sequenceto RNA. Typically, the promoter refers to a DNA segment which binds witha polymerase and transcription factors, and the enhancer refers to a DNAsegment which binds with activation domains of the polymerase.

The term “untranslated region (UTR)” as used herein is a sequencelocated at the 3′- and 5′-ends of the coding sequence, and it contains apolyadenylation signal as a transcription termination region, and thelike.

The term “intron” as used herein is an untranslated nucleotide sequencelocated between exons translated to a protein after transcription. Thetranscribed mRNA precursor is converted into a mature mRNA after theintrons are removed through splicing. The term “intron” as used hereinis also intended to comprehend a splicing donor, a splicing acceptor, atriple guanine repeat sequence (G-triple motif), and/or a branchsequence.

The term “locus control region (LCR)” as used herein is a nucleotidesequence having a DNase I-sensitive site. Tissue-specific transcriptionfactors are bound to LCRs.

The term “operably linked” as used herein means the association of oneor more nucleic acid sequences coupled to a single nucleic acid fragmentsuch that the function of the single fragment is affected. For example,a promoter is operably linked with a coding sequence to enhance theexpression of the coding sequence.

The present invention is described in detail hereinafter.

As described above, for effective gene therapy, a target gene should bespecifically expressed in a specific tissue or cell in a sustainedmanner so that a pathogenic gene is replaced by the target gene. Fortissue-specific and sustained gene expression, such an expression vectoris required to include improved transcriptional regulatory elements.

Accordingly, the present invention provides a truncated FVII (FVIIΔ)promoter having the nucleotide sequence as set forth in SEQ ID NO: 42which results from the truncation of a 207 bp fragment upstream of the5′-end of a 504 bp FVII promoter by deleting XhoI (5′-end) and BamHI(3′-end) restriction sites from the nucleotide sequence of SEQ ID NO:41. The following examples show that the FVIIΔ promoter has an activitywhich is 2.5-fold or more higher than that of the untruncated FVIIpromoter.

The FVIIΔ promoter may be combined with a suitable enhancer, e.g., atleast one selected from the group consisting of a PAH enhancer havingthe nucleotide sequence as set forth in SEQ ID NO: 44 and an AMBPenhancer having the nucleotide sequence as set forth in SEQ ID NO: 45.

The present invention also provides an intron selected from the groupconsisting of polynucleotides having the nucleotide sequences as setforth in SEQ ID NOS: 46 to 57.

The introns of SEQ ID NOS: 46, 47 and 48 result from the truncation ofintrons 1 of human antithrombin, plasminogen and prothrombin,respectively. The introns of SEQ ID NOS: 49, 50 and 51 are syntheticintrons that include the nucleotide sequences of SEQ ID NOS: 46, 47 and48, respectively, and share a splicing donor sequence, a triple guaninerepeat sequence (G-triple motif) derived from intron 2 of humanα-globin, a branch sequence of human plasminogen, and a consensussplicing acceptor sequence. The introns of SEQ ID NOS: 52, 53 and 54 aresynthetic introns lacking the G-triple motif and the branch sequence ofthe nucleotide sequences of SEQ ID NOS: 49, 50 and 51, respectively. Theintron of SEQ ID NO: 55 is a synthetic intron that includes thefull-length intron 1 of antithrombin, a splicing donor sequence and aconsensus splicing acceptor sequence, and the introns of SEQ ID NOS: 56and 57 are synthetic introns that result from the truncation of thenucleotide sequence of SEQ ID NO: 55. Among the above, the intron of SEQID NO: 57 is more preferred.

The present invention also provides a locus control region (LCR)selected from the group consisting of polynucleotides having thenucleotide sequences as set forth in SEQ ID NOS: 58 to 61.

The LCRs of SEQ ID NOS: 58, 59, 60 and 61 are located at −108 bp and−7.8 kb sites upstream of human α1-antitrypsin gene, −3.8 kb siteupstream of human α-fetoprotein, and −6 kb site upstream of humanalbumin gene, respectively, among which the LCR of SEQ ID NO: 58 ispreferred.

Polynucleotides which are substantially the same as, and functionallysimilar to, the sequences mentioned in the present invention are alsowithin the scope of the present invention. The term “substantially thesame and functionally similar polynucleotide” means that onepolynucleotide has a nucleotide sequence which is at least 70%,preferably 80%, more preferably 90% and the most preferably 95%identical to the other polynucleotide, wherein the identity isdetermined by computerized algorithm or software.

The present invention also provides an expression vector including atranscriptional regulatory element and a coding sequence operably linkedto and under control of the transcriptional regulatory element, whereinthe transcriptional regulatory element includes:

1) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42;

2) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42 andat least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 44 and 45operably linked to the polynucleotide of SEQ ID NO: 42;

3) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42 andat least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to 57operably linked to the polynucleotide of SEQ ID NO: 42;

4) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42 andat least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to 61operably linked to the polynucleotide of SEQ ID NO: 42;

5) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42; atleast one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 44 and45; and at least one polynucleotide selected from the group consistingof polynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to57, said polynucleotides being operably linked to each other;

6) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42; atleast one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 44 and45; and at least one polynucleotide selected from the group consistingof polynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to61, said polynucleotides being operably linked to each other;

7) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42; atleast one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to 57;and at least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to 61,said polynucleotides being operably linked to each other; or

8) a polynucleotide having the nucleotide sequence of SEQ ID NO: 42; atleast one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 44 and45; at least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 46 to 57;and at least one polynucleotide selected from the group consisting ofpolynucleotides having the nucleotide sequences of SEQ ID NOS: 58 to 61,said polynucleotides being operably linked to each other.

The present invention also provides an expression vector including atranscriptional regulatory element and a coding sequence operably linkedto and under control of the transcriptional regulatory element, thetranscriptional regulatory element including a promoter and apolynucleotide which is operably linked to the promoter and selectedfrom the group consisting of: at least one polynucleotide selected fromthe group consisting of polynucleotides having the nucleotide sequencesof SEQ ID NOS: 46 to 57; at least one polynucleotide selected from thegroup consisting of polynucleotides having the nucleotide sequences ofSEQ ID NOS: 58 to 61; and at least one polynucleotide selected from thegroup consisting of polynucleotides having the nucleotide sequences ofSEQ ID NOS: 46 to 57 operably linked to at least one polynucleotideselected from the group consisting of polynucleotides having thenucleotide sequences of SEQ ID NOS: 58 to 61.

Preferably, the expression vector may further include polynucleotideshaving the nucleotide sequences of SEQ ID NOS: 62 and 63 at the 5′- and3′-ends of the coding sequence. The nucleotide sequences of SEQ ID NOS:62 and 63 may be derived from 5′ and 3′ UTRs of FIX gene.

The coding sequence may be selected from the nucleotide sequencesencoding liver-specific proteins including, but not limited to, albumin,α-fetoprotein, α-glucosidase, α1-antitrypsin, antithrombin,lipoproteins, ceruloplasmin, FVII, FVIII, FIX, erythropoietin,fibrinogen, glucocerebrosidase, haptoglobin, IGF-1, insulin,plasminogen, prothrombin, and transferrin.

The coding sequence is operably and controllably linked to a promoter,an enhancer, an intron, a UTR and an LCR.

As described above, an appropriate combination of the transcriptionalregulatory elements of the present invention enables the livertissue-specific expression of a target coding sequence, and contributesto mRNA stabilization, thereby resulting in improved expression of thecoding sequence. An expression cassette or vector including variouscombinations of such transcriptional regulatory elements enablessustained expression of a target coding sequence in a liver tissue at ahigh level, and thus, can be broadly applied for the treatment ofthrombosis, hemophilia, liver cancer, etc.

The following Examples are intended to illustrate the present inventionwithout limiting its scope.

Example 1 Isolation and Activity Analysis of Liver Tissue-SpecificExpression Promoters and Enhancers <Step 1> Isolation and ActivityAnalysis of Liver Tissue-Specific Expression Promoters

Genomic DNA was extracted from a cell lysate of human liver cell line(Chang cells) by using a DNeasy Tissue Kit (Qiagen). In order to isolatea FVII promoter, PCR was performed using the genomic DNA as a template,a primer set of SEQ ID NOS: 1 and 2, and DNA polymerase (Ex-Taq, Takara)to obtain a DNA fragment having the nucleotide sequence of SEQ ID NO:41. Specifically, PCR was carried out under the following conditions:initial denaturation at 94° C. for five minutes; 30 cycles ofdenaturation at 94° C. for 30 seconds, annealing at 56° C. for 30seconds and extension at 72° C. for one minute; and final extension at72° C. for three minutes. The PCR products were purified by gelextraction and inserted into a pCR2.1-TOPO plasmid vector. The FVIIpromoter having the nucleotide sequence of SEQ ID NO: 41 was identifiedby a restriction enzyme cleavage map and sequence analysis. The plasmidcontaining the FVII promoter was designated “pCR-FVII pro.” Further, PCRwas carried out as described above except for using a primer set (SEQ IDNOS: 3 and 4) for an OATP-C promoter and a primer set (SEQ ID NOS: 5 and6) for an AAT promoter, and the PCR products were inserted intopCR2.1-TOPO plasmid vectors. The OATP-C promoter having the nucleotidesequence of SEQ ID NO: 43 and the AAT promoter were identified by arestriction enzyme cleavage map and sequence analysis. The plasmidscontaining the OATP-C promoter and the AAT promoter were designated“pCR-OATP-C pro” and “pCR-AAT enh/pro,” respectively. Schematic diagramsof pCR-FVII pro, pCR-OATP-C pro and pCR-AAT enh/pro are shown in FIG.1A.

SV40 late poly(A) of a phRL-null vector (Promega) expressing Renillaluciferase was replaced with poly(A) of human growth hormone, and a T7promoter and introns were removed therefrom to construct a pmRL-nullvector. The pmRL-null vector was digested with BglII restriction enzymeand treated with a shrimp alkaline phosphatase.

The pCR-FVII pro and pCR-OATP-C pro plasmids were digested with BamHI.The obtained DNA fragments were purified by gel extraction and insertedinto the previously prepared pmRL-null vectors. The desired DNA fragmentwas identified in each plasmid vector by a restriction enzyme cleavagemap. The plasmid vectors were designated “pmRL-FVII Pro” and“pmRL-OATP-C Pro.”

Meanwhile, an about 200 bp fragment of the 5′-end of the FVII promoterwithin the pmRL-FVII Pro was truncated using EcoRI restriction enzyme,followed by ligation to construct a pmRL-FVIIΔ Pro plasmid carrying atruncated FVII (FVIIΔ) promoter having the nucleotide sequence of SEQ IDNO: 42.

Schematic diagrams of pmRL-FVII Pro, pmRL-OATP-C Pro and pmRL-FVIIΔ Proare shown in FIG. 1B.

Luciferase expression levels of the pmRL-FVII Pro, pmRL-FVIIΔ Pro andpmRL-OATP-C Pro were measured in human liver cell lines (Huh-7, HepG2,Hep3B), kidney cell line (HEK293), lung cancer cell line (A549) andcervical cancer cell line (HeLa). In detail, the cells were cultured ineach well of a six-well plate using a polyethyleneimine (PEI) reagent(Polyplus, Illkirch, France) to reach 70 to 80% confluency. The culturedcells were transfected with each 2 μg of the pmRL-FVII Pro, pmRL-FVIIΔPro and pmRL-OATP-C Pro plasmids and 1 μg of a pcDNA-lacZ plasmid andcultured at 37° C. for 24 hours. The cells were harvested andcentrifuged at 3,000 rpm for five minutes to separate cells and media.The cells were resuspended in 100 μl of a lysis buffer (25 mMTris-phosphate, pH 7.8, 2 mM DTT (Dithiothreitol), 2 mM1,2-diaminocyclohexane N,N,N,N′-tetra acetic acid, 10% glycerol, 1%Triton® X-100) followed by freezing and thawing (×3) to completely lysethe cells. The cell lysates were centrifuged at 10,000 rpm for oneminute. The resultant supernatants were subjected to galactosidase assayand Bradford assay to normalize for transfection efficiency, andpromoter activity was measured by luciferase activity assay. The resultsare shown in FIG. 2A.

As shown in FIG. 2A, the FVII promoter and OATP-C promoter induced ahigher luciferase expression specifically in the liver cell line Huh-7.

The FVIIΔ promoter showed a 2.5-fold higher activity than the FVIIpromoter in the Huh-7 cells (see FIG. 2B).

The FVIIΔ promoter showed 1.4% of the expression efficiency of a CMVpromoter in the Huh-7 cells, and average 0.07% of the expressionefficiency of the CMV promoter in the cell lines derived from othertissues than a liver tissue. However, unlike the CMV promoter, thespecificity of the FVIIΔ promoter to the liver tissue was 20-fold higherthan other tissues (see FIG. 2C). This result shows that the FVIIΔpromoter increases the expression efficiency of a target gene withoutadversely affecting the liver tissue specificity, and thus, is suitablefor an expression vector which can be selectively operated in the livertissue. In FIG. 2C, (−) represents a negative control (absence of apromoter), ICAM2 represents a promoter of an intracellular adhesionmolecule, Tie2 represents a promoter of a tyrosine kinase receptor,Endoglin represents a promoter of endoglin, and HBcP represent apromoter of a hepatitis B virus core protein.

<Step 2> Isolation of Enhancers

In order to isolate a PAH enhancer, PCR was performed as described in<step 1> except for using a primer set (SEQ ID NOS: 7 and 8) for the PAHenhancer. The PCR product was purified by gel extraction and insertedinto a pCR2.1-TOPO plasmid vector. The PAH enhancer having thenucleotide sequence of SEQ ID NO: 44 was identified by a restrictionenzyme cleavage map and sequence analysis. The plasmid was designated“pCR-PAHenh.” Further, the above procedure was repeated except for usinga primer set (SEQ ID NOS: 9 and 10) for an AMBP enhancer to construct apCR-AMBPenh plasmid carrying the AMBP enhancer having the nucleotidesequence of SEQ ID NO: 45. Schematic diagrams of the pCR-PAHenh andpCR-AMBPenh plasmids are shown in FIG. 3A.

The PAH and AMBP enhancers were isolated by digesting the plasmidspCR-PAHenh and pCR-AMBPenh with SpeI and XbaI, purified by gelextraction, and inserted into the SpeI sites of the pmRL-FVIIΔPro andpmRL-OATP-C Pro vectors prepared in <step 1>. The pmRL-FVIIΔ Pro andpmRL-OATP-C Pro combined with the PAH enhancer were designated “pmRL-PF”and “pmRL-PO,” respectively. The pmRL-FVIIΔ Pro and pmRL-OATP-C Procombined with the AMBP enhancer were designated “pmRL-AF” and “pmRL-AO,”respectively. The PAH enhancer was extracted from the pmRL-PF by usingSpeI and XbaI, and inserted into the SpeI sites of the pmRL-AF andpmRL-AO. The resultant vectors were designated “pmRL-PAF” and“pmRL-PAO.” Schematic diagrams of the pmRL-PF, pmRL-PO, pmRL-AF,pmRL-AO, pmRL-PAF and pmRL-PAO vectors are shown in FIG. 3B.

CMV and SV40 enhancers, which can improve promoter activity in atissue-nonspecific manner, were inserted into upstream of the OATP-C andFVII promoters according to the above-described procedure.

<Step 3> Analysis of Expression Efficiency of Target Gene According toCombination of Promoter and Enhancer <3-1> In Vitro Test

The gene expression efficiencies for the vectors constructed in <step 2>were measured in a human liver cell line (Huh-7) and control cell lines,i.e., a human lung cell line (A549) and a human cervical cancer cellline (HeLa), in the same manner as in <step 1>. The results are shown inFIG. 4A.

As shown in FIG. 4A, the specificity of the vector carrying the PAHenhancer and FVIIΔ promoter in the liver cell line Huh-7 was average28.4-fold higher than that in the other cell lines. This shows that thetissue-specificity of the FVIIΔ promoter was increased 1.42-fold by thePAH enhancer. The tissue-specificity of the FVIIΔ promoter was increased213.7-fold or more by the AMBP enhancer, which is about 3-fold higherthan an increase (65.6-fold) of the tissue-specificity of the OATP-Cpromoter by the AMBP enhancer. The results show that liver-selectivityof the FVIIΔ promoter can be significantly enhanced by an enhancer.

In order to measure the activity of the expression vector controlledunder the AMBP enhancer and the FVIIΔ promoter relative to that of theexpression vector controlled under the CMV promoter, a luciferaseexpression level was measured in human liver cell lines (Huh-7, Hep3B)and control cell lines, i.e., a kidney cell line (HEK293), a lung cancercell line (A549), a cervical cancer cell line (HeLa) and a vascularendothelial cell line (HUVEC). The results are shown in FIG. 4B.

As shown in FIG. 4B, in the liver cell lines, the expression vectorcontaining the AMBP enhancer and the FVIIΔ promoter showed 8.7% of theluciferase expression efficiency of the expression vector containing theCMV promoter, and in the other cell lines, average 0.14% of theluciferase expression efficiency of the expression vector containing theCMV promoter. This shows that the liver tissue-specificity of theexpression vector containing the AMBP enhancer and the FVIIΔ promoter isabout 62-fold higher than the other tissues.

<3-2> In Vivo Test

In order to measure the in vivo gene expression efficiency in the liver,each complex of the pmRL plasmids of <step 2> with polyethyleneimine(PEI) (Polyplus, Illkirch, France) or in vivo jetPEI (Polyplus,Illkirch, France) was injected into a mouse tail vein. Two days later,the liver was extracted from the mouse, and a luciferase expressionlevel was measured.

In detail, each 40 μg of the pmRL-PO, -PF, -AO and -AF plasmids and 10μg of a pcDNA-LacZ plasmid were injected into the tail veins of mice(six weeks old) in the form of a complex with PEI or In vivo jetPEI in a5% glucose solution. Two days later, the liver, kidney, heart, spleenand lung tissues were extracted from the mice and homogenized in a PBSsolution with a homogenizer. Then, 500 μl of the resultant tissuesolution was mixed with 500 μl of a 2× lysis buffer (50 mMTris-phosphate, pH 7.8, 4 mM DTT (Dithiothreitol), 4 mM1,2-diaminocyclohexane N,N,N,N′-tetra acetic acid, 20% glycerol, 2%Triton® X-100) followed by freezing and thawing (×3) to completely lysethe cells. The cell lysates were centrifuged at 10,000 rpm for oneminute. The resultant supernatants were subjected to galactosidase assayand Bradford assay to normalize for transfection efficiency, and geneexpression efficiency was measured by luciferase activity assay. Theresults are shown in FIG. 4C.

As shown in FIG. 4C, in the liver tissue, a luciferase expression levelinduced by the complex of the expression vector containing FVIIΔpromoter/PAH enhancer and PEI was equal to 14% of that induced by theCMV promoter, which is 14-fold or more higher than that of in vitroassay of luciferase expression. A liver-specific luciferase expressionlevel induced by FVIIΔ promoter/AMBP enhancer was equal to 80% of thatinduced by the CMV promoter, which is 10-fold or more higher than thatof in vitro assay of luciferase expression.

Using in vivo jetPEI instead of PEI increased the gene expressionefficiency in the liver. Specifically, a liver-specific luciferaseexpression level induced by the complex of the expression vectorcontaining OATP-C promoter/PAH enhancer and in vivo jetPEI was equal toabout 38% of that induced by the CMV promoter, and a luciferaseexpression level induced by FVIIΔ promoter/PAH enhancer was equal toabout 80% of that induced by the CMV promoter. The liver-specificluciferase expression level induced by the complex of FVIIΔpromoter/AMBP enhancer and in vivo jetPEI was similar to that induced bythe CMV promoter. These results show that the activities of theinventive promoters and enhancers are better in vivo than in vitro.

Example 2 Isolation of UTRs of hFIX and Introns of Liver-Specific Genes,and Analysis of Gene Expression Efficiency by UTRs and Introns

<Step 1> Identification of UTRs of hFIX, and Construction of ExpressionVectors Including UTRs

1.2 mg of total RNA was extracted from 1 g of a human liver tissue,which was previously homogenized with a homogenizer, using a RNAextraction kit (Pharmacia Biotech). A single-stranded DNA wassynthesized using the extracted RNA as a template, an M-MuLV reversetranscriptase (Takara) and oligo-dT17 primers (Takara), and adouble-stranded cDNA was synthesized from the single-stranded DNA usingDNA polymerase I (Takara).

In order to evaluate the effect of FIX UTR on gene expressionefficiency, PCR was performed using the cDNA as a template, a primer set(SEQ ID NOS: 11 and 12) designed from a nucleotide sequence of FIX(Genbank accession No: NM000133) and Ex-Taq to obtain FIX cDNA including5′UTR (SEQ ID NO: 62) and 3′UTR (SEQ ID NO: 63). The PCR condition wasas follows: initial denaturation at 94° C. for five minutes; 30 cyclesof denaturation at 94° C. for one minute, annealing at 56° C. for oneminute and extension at 72° C. for one minute and 30 seconds; and finalextension at 72° C. for three minutes.

The above procedure was repeated except for using a primer set of SEQ IDNOS: 13 and 14 to obtain FIX cDNA including no UTR as a control.

The amplified DNA fragments were digested with restriction enzymes NotIand SalI, purified by gel extraction, and inserted into NotI and SalIrestriction sites of pBluescript SK(+) vectors. The FIX including thedesired UTRs and the FIX including no UTR were identified by arestriction enzyme cleavage map and sequence analysis. The vectors weredesignated “pBS-hFIXUTR” and “pBS-hFIX,” respectively (see FIG. 5( a)).

<Step 2> Isolation of Introns of Liver-Specific Genes and Constructionof Expression Vectors Including the Introns

<2-1> Construction of pCR-int

First, PCR was performed to obtain fragments including about 300 bp of5′-ends of introns 1 of human antithrombin, plasminogen and prothrombin.

In detail, PCR was performed using the genomic DNA described in <step 1>of Example 1 as a template, and primer sets for human antithrombinintron (SEQ ID NOS: 15 and 16), human plasminogen intron (SEQ ID NOS: 17and 18) and human prothrombin intron (SEQ ID NOS: 19 and 20) under thefollowing conditions: initial denaturation at 94° C. for five minutes;30 cycles of denaturation at 94° C. for one minute, annealing at 60° C.for one minute and extension at 72° C. for two minutes and 30 seconds;and final extension at 72° C. for three minutes. The PCR products werepurified by gel extraction and inserted into pCR2.1-TOPO vectors. Theintrons of SEQ ID NOS: 46, 47 and 48 were identified by a restrictionenzyme cleavage map and sequence analysis. The plasmids were designated“pCR-ATint,” “pCR-PLAint” and “pCR-PTint,” respectively.

Further, in order to isolate the intron 1 of FIX reported by Kurachi S,the above PCR procedure was repeated except for using a primer set (SEQID NOS: 21 and 22) for the intron 1 of FIX, and the obtained PCR productwas inserted into a pCR2.1-TOPO vector. The resulting plasmid vector wasdigested with restriction enzyme PvuI or ScaI, and self-ligated. The FIXintrons having different sizes were identified by a restriction enzymecleavage map and sequence analysis. The plasmids were designated“pCR-1.4kbFIXint” and “pCR-0.3kbFIXint,” respectively.

<2-2> Construction of pBS-FIX-Syn1int

The intron fragments of the pCR-ATint, pCR-PLAint and pCR-PTint preparedin <2-1> were inserted between exons 1 and 2 of FIX.

In detail, PCR of <2-1> was repeated except for using a sense primer(SEQ ID NO: 11) for FIX 5′UTR and an antisense primer (SEQ ID NO: 23)for consensus splicing donor sequence GTAA and G-triple motif derivedfrom intron 2 of human α-globin to obtain a DNA fragment including FIX5′UTR and exon 1, splicing donor and human α-globin G-triple motif.Further, the above PCR was repeated except for using a sense primer (SEQID NO: 24) for the branch sequence of human plasminogen and consensussplicing acceptor sequence TCGA and an antisense primer (SEQ ID NO: 12)for FIX 3′UTR to obtain a DNA fragment including the branch sequence,splicing acceptor, exons 2 to 8 and 3′UTR of FIX. The PCR products werepurified by gel extraction and inserted into pCR2.1-TOPO vectors. Thedesired DNA fragments were identified by a restriction enzyme cleavagemap and sequence analysis. The plasmids were designated ‘pCR-5′hFIX” and“pCR-3′ hFIX,” respectively.

The pCR-5′ hFIX and pCR-3′ hFIX were digested with restriction enzymesNotI and NdeI, and NdeI and SalI, respectively, and both weresimultaneously inserted into NotI and SalI restriction sites ofpBluescript. The resulting plasmids were digested with NdeI, and thepCR-ATint, pCR-PLAint and pCR-PTint of <2-1> pretreated with the sameenzyme, NdeI were inserted thereto. The desired introns of SEQ ID NOS:49, 50 and 51 were identified by a restriction enzyme cleavage map andsequence analysis. The plasmids were designated “pBS-hFIXUTR-Syn1AT,”“pBS-hFIXUTR-Syn1PLA” and “pBS-hFIXUTR-Syn1PT,” respectively. Schematicdiagrams of the plasmids are shown in FIG. 5 (b).

<2-3> Construction of pBS-FIX-Syn2int

The pBS-FIXUTR-Syn1PLA plasmid of <2-2> was digested with XhoI and AatIIfor the XhoI and AatII restriction sites adjacent to the splicing donorand acceptor, and the pCR-ATint, pCR-PLAint, pCR-PTint, pCR-1.4kbFIXintand pCR-0.3kbFIXint of <2-1> pretreated with the same enzymes, XhoI andAatII were inserted thereto. The introns of Syn2AT, Syn2PLA and Syn2PT(SEQ ID NOS: 52, 53 and 54) and introns of 1.4kbFIXint and 0.3kbFIXintwere identified by a restriction enzyme cleavage map. The plasmids weredesignated “pBS-hFIXUTR-Syn2AT,” “pBS-hFIXUTR-Syn2PLA,”“pBS-hFIXUTR-Syn2PT,” “pBS-hFIXUTR-1.4kbFIXint (hFIXm1)” and“pBS-hFIXUTR-0.3kbFIXint (hFIXm2),” respectively. Schematic diagrams ofthe plasmids are shown in FIG. 5 (c).

<2-4> Isolation of Full-Length Intron 1 of Antithrombin and Constructionof Shortened Antithrombin Introns

The PCR of <2-1> was repeated except for using a primer set of SEQ IDNOS: 15 and 25 to amplify a fragment including full-length intron 1 ofantithrombin. About 2.3 kb PCR products were inserted into pCR2.1-TOPOvectors, which were designated “pCR-NAint.” The resulting plasmids weredigested with XhoI and AatII, and inserted into pBS-hFIXUTR according tothe procedure of <2-3> to obtain pBS-hFIXUTR-NA including the intron ofSEQ ID NO: 55.

The intron inserted into pBS-hFIXUTR-NA was truncated from PvuII siteusing a Kilo-Sequence Deletion Kit (Takara). Specifically, pBS-FIXUTR-NAwas digested with PvuII, degraded with exonuclease III for 15 sec, 30sec, 45 sec and 1 min at 25° C., end-blunted with Mung Bean nuclease andklenow enzyme, and self-ligated. The resultant constructs weretransformed into E. coli. The obtained transformants were cultured in anampicilin plate, single colonies were selected therefrom, and the sizesof the introns of the plasmids derived from the colonies were determinedby electrophoresis using restriction enzymes. The plasmids carryingproper-sized introns were purified and sequenced to obtain the plasmidspBS-hFIXUTR-ΔNAL including a 811 bp intron of SEQ ID NO: 56 andpBS-hFIXUTR-ΔNAS including a 640 bp intron of SEQ ID NO: 57. Schematicdiagrams of the plasmids pBS-hFIXUTR-NA, pBS-hFIXUTR-ΔNAL andpBS-hFIXUTR-ΔNAS are shown in FIG. 5 (d).

<Step 3> Analysis of Effects of UTRs and Introns on FIX Expression

The pBS-hFIX, pBS-hFIXUTR, pBS-hFIXUTR-Syn1int (AT, PLA, PT) andpBS-hFIXUTR-Syn2int (AT, PLA, PT, 1.4kbFIXint, 0.3kbFIXint) plasmidsprepared in <step 1> and <step 2> were digested with NotI and SalI, andinserted into pTRUF6 adeno-associated virus vectors containing a CMVpromoter and bovine poly(A) to obtain hFIX expression vectors, CMV-hFIX,CMV-hFIXUTR, CMV-hFIXUTR-Syn1int (AT, PLA, PT) and CMV-hFIXUTR-Syn2int(AT, PLA, PT, 1.4kbFIXint, 0.3kbFIXint).

25 μg of the plasmid DNAs of the above expression vectors were injectedinto C57BL/6 mouse tail veins. The next day, blood samples werecollected and used in the following ELISA.

The obtained blood samples were diluted 100 times in a HBS-BSA-EDTA-T20buffer (0.1M HEPES-0.1M NaCl-1% BSA-10 mM EDTA-0.1% Tween-20). Thediluted blood samples were added to a 96-well plate coated with humanFIX (hFIX) antibody (Affinity Biologicals), cultured at room temperaturefor 90 minutes, washed with PBS-0.1% Tween-20, and incubated with asecondary antibody labeled with peroxidase. The plate was washed againwith PBS-0.1% Tween-20, and a substrate buffer containing dissolvedO-phenyldiamine and H₂O₂ was added thereto. The plate was incubated forfive minutes, and the reaction was stopped with 2.5M H₂SO₄. Theabsorbance was measured at 490 nm with a spectrophotometer (SpectraShell Microplate Reader, STL Spectra, Milan, Italy), and hFIXconcentrations of the samples were calculated according to a standardcurve of a standard absorbance versus a standard concentration. Theresults are shown in FIG. 6A.

As shown in FIG. 6A, the day after injection, blood concentration ofhFIX protein induced by CMV-hFIXUTR was about 910 ng/ml that is about1.7-fold higher than that (540 ng/ml) of the protein induced by CMV-hFIXincluding no UTR. This shows that the UTR contributes to the expressionof hFIX.

The hFIX expression level (1,480 ng/ml) of CMV-hFIXUTR-Syn1PLA was up to1.6-fold of that (910 ng/ml) of CMV-hFIXUTR, and the hFIX expressionlevel (2,170 ng/ml) of CMV-hFIXUTR-Syn2AT was up to 2.4-fold of that(910 ng/ml)) of CMV-hFIXUTR. This shows that the intron is involved inhFIX gene expression and initial hFIX protein induction.

Meanwhile, recombinant adeno-associated viruses rAAV-CMV-hFIX,rAAV-CMV-hFIXUTR and rAAV-CMV-hFIXUTR-Syn1int (AT, PLA, PT) wereproduced using the expression vectors CMV-hFIX, CMV-hFIXUTR andCMV-hFIXUTR-Syn1int (AT, PLA, PT). Specifically, 200 ml of HEK293Tcells, which were adapted to a concentration of 1×10⁶ cells/ml using lowcalcium DMEM medium (0.1 mM Ca⁺⁺, 0.1% PL-68, 1% FBS), were prepared ina 500 ml Spinner flask. A DNA-PEI mixture including 33 μg of eachexpression vector, 167 μg of adenovirus helper plasmid pDG and 650 μl of10 μM PEI was loaded on the cells, and the cells weresuspension-cultured in a 5% CO₂ incubator at 30 rpm for six hours. Then,the culture medium was replaced with 100 μg dextran sulfate-low calciumDMEM. After 48-hour culture, the cells were harvested, subjected tofreezing and thawing (×3) and centrifuged at 2000 rpm for 5 minutes. Theresultant supernatants were purified by iodixanol gradientultracentrifugation (Zolotukhin, S. et al, Gene Ther., 6: 973-985(1999)). 1×10⁹ infectious particles (IP) were injected into tail veinsof immunodeficient nude mice (Japan SLC Inc). After two weeks, bloodsamples were collected every week and subjected to ELISA according tothe above-described method. The results are shown in FIG. 6B.

As shown in FIG. 6B, at 14 weeks after virus injection, the hFIXexpression levels of the viruses including the inventive introns were 10to 20-fold higher than that of the virus including no intron.Specifically, the hFIX protein concentration of the virus carryingCMV-hFIXUTR-Syn1AT was 19,996 pg/ml, while the hFIX proteinconcentration of the virus carrying CMV-hFIXUTR was 2,024 pg/ml. Thisshows that the inventive introns play critical roles in enhanced geneexpression.

<Step 4> Analysis of Luciferase Expression Efficiency and mRNA Stabilityby Introns<4-1> Introduction of Intron into Luciferase Expression VectorControlled by TK Promoter

A pRL-TK expression vector (promega) was digested with HindIII/NheI toremove a SV40 intron, blunt-ended and self-ligated. The intron-truncatedpRL-TK vector was designated “pRL-Δint” and the SV40 intron-containingpRL-TK vector was designated “pRL-SV40.”

The pBS-hFIXUTR-Syn1AT, pBS-hFIXUTR-Syn1PLA and pBS-hFIXUTR-0.3kbFIXintobtained in <2-2> and <2-3> were digested with XhoI/AatII andblunt-ended. The resultant fragments were inserted into the aboveexpression vector pRL-Δint. The resulting vectors were designated“pRL-Syn1AT,” “pRL-Syn1PLA” and “pRL-hFIXm2,” respectively (see FIG.7A).

<4-2> Analysis of Luciferase Expression Efficiency

A complex of 2 μg of the luciferase expression vectors of <4-1>, 1 μg ofa β-galactosidase expression vector and 6 μg of PEI (polyplus) wasinjected into liver cell line (Hep3B), kidney cell line (HEK293) andlung cell line (A549) as described in <step 1> of Example 11. After 48hours, the cells were harvested and luciferase expression efficiency wasmeasured according to the same method as in <step 1> of Example 1. Theresults are shown in FIG. 7B.

As shown in FIG. 7B, the luciferase expression induced by theintron-containing expression vectors was up to 200-fold higher than thatinduced by the intron-free expression vectors (in case of Syn1AT inHep3B liver cell line). Particularly, the Syn1AT expression vectorsignificantly contributed to the induction of luciferase activity aswell as the induction of FIX expression as described in <step 3>.

<4-3> Analysis of mRNA Expression Stability

The luciferase expression vectors of <4-1> were transfected into A549lung cell line according to the same method as in <4-2>, and the cellswere harvested after 48 hours. Total RNA was extracted from the cellsusing a FastPure RNA kit (Takara). Single-stranded. DNAs were preparedusing 500 ng of each total RNA as a template and AMV RTase (Takara).Then, PCR was performed using the above single-stranded DNAs as atemplate, and primer sets for detecting luciferase and β-actin. Theresults are shown in FIGS. 8A and 8B.

As shown in FIGS. 8A and 8B, the relative amount of RLuc mRNA induced bythe pRL-Syn1AT vector was 1.7-fold higher than that of RLuc mRNA inducedby the pRL-Δint vector. It shows that the inventive introns areresponsible for higher stability of mRNA, which leads to enhanced geneexpression.

<Step 5> Analysis of Effects of Antithrombin Introns on FIX Expression

In order to determine the effects of antithrombin introns on hFIXprotein expression, the pBS-hFIXUTR-NA, pBS-hFIXUTR-ΔNAL andpBS-hFIXUTR-NAS plasmids prepared in <2-4> were inserted into pTRUF6adeno-associated virus vectors according to the same method as in <step3> to obtain expression vectors CMV-hFIXUTR-NA, CMV-hFIXUTR-ΔNAL andCMV-hFIXUTR-ΔNAS. Plasmid DNAs of the expression vectors CMV-hFIX,CMV-hFIXUTR, CMV-hFIXUTR-0.3kbFIXint (CMV-hFIXm2), CMV-hFIXUTR-Syn1AT,CMV-hFIXUTR-Syn2AT, CMV-hFIXUTR-ΔNA, CMV-hFIXUTR-ΔNAL andCMV-hFIXUTR-ΔNAS were injected into mouse tail veins according to thesame method as in <step 3>, and blood hFIX concentrations in subjectswere measured by ELISA. The results are shown in FIG. 9A.

As shown in FIG. 9A, hFIX protein concentrations of expression vectorscontaining the synthetic or native antithrombin intron were 1.7 to3.5-fold higher than that of CMV-hFIXUTR (1,730 to 3,540 ng/ml versus1,000 ng/ml). Particularly, the CMV-hFIXUTR-ΔNAL expression vectorshowed about 1.7-fold higher expression efficiency than the previouslyestablished CMV-hFIXm2 expression vector (2,010 ng/ml versus 1,000ng/ml). This result shows that the ΔNAL intron is very suitable for hFIXoverexpression.

Meanwhile, in order to evaluate the effect of the intron ΔNAL on hFIXexpression in vitro, the expression vectors CMV-hFIX, CMV-hFIXUTR andCMV-hFIXUTR-ΔNAL were transfected into HEK293T kidney cell line. At twodays after the transfection, the cells together with media werecollected and lysed. The hFIX protein level in the lysates was measuredby electrophoresis using an hFIX antibody and the results are shown inFIG. 9B.

As shown in FIG. 9B, the CMV-hFIXUTR-ΔNAL expression vector induced1.55-fold higher hFIX expression level than the CMV-hFIX and CMV-hFIXUTRexpression vectors. This result shows that the ΔNAL intron is efficientfor hFIX overexpression.

Example 3 Isolation of Liver-Specific LCRs and Analysis of GeneExpression Efficiency by LCRs <Step 1> Isolation of Liver-Specific LCRsand Construction of Plasmid Vectors Including LCRs

The positions of the TGTTTGC motif was analyzed in the downstream ofα1-antitrypsin, α-fetoprotein and albumin genes expressed only in theliver tissues. The results are shown in FIG. 10.

As shown in FIG. 10, seven TGTTTGC motifs are distributed at −7.8 kbsite of α1-antitrypsin gene, six motifs being in a forward orientationand one motif being in a reverse orientation, and one motif is locatedin a forward orientation at −108 bp site of α1-antitrypsin gene. At −3.8kb site of α-fetoprotein, two TGTTTGC motifs are distributed in aforward orientation and one motif is in a reverse orientation and at −6kb site of albumin gene, one motif is located in a reverse orientation.In addition, TGTTTGC motifs are clustered at −800 bp site of theα1-antitrypsin gene, −1.5 kb and −500 bp sites of the α-fetoproteingene, and −10 kb, −3.5 kb and −2.6 kb sites of the albumin gene. Amongthese sites, −7.8 kb and −108 bp sites of the α1-antitrypsin gene, −3.8kb site of the α-fetoprotein gene and −6 kb site of the albumin genewere isolated.

In detail, in order to isolate LCR located at −108 bp site of theα1-antitrypsin gene, PCR was performed using the genomic DNA of <step 1>of Example 1 as a template, a primer set of SEQ ID NOS: 26 and 27, andDNA polymerase (Ex-Taq, Takara) according to the same method as in <step1> of Example 1. The PCR products were purified by gel extraction, andinserted into pCR2.1-TOPO vectors. The desired α1-antitrypsin LCR havingthe nucleotide sequence of SEQ ID NO: 58 was identified by a restrictionenzyme cleavage map and sequence analysis. The plasmid was designated“pCR-AAT108lcr.”

Similarly, the above PCR procedure was repeated except for using primersets of SEQ ID NOS: 28 and 29, SEQ ID NOS: 30 and 31, and SEQ ID NOS: 32and 33 to amplify LCRs located at −7.8 kb site of the humanα1-antitrypsin, −3.8 kb site of the human α-fetoprotein and −6 kb siteof the human albumin gene, respectively. The PCR products were insertedinto pCR2.1-TOPO vectors. The desired LCRs having the nucleotidesequences of SEQ ID NOS: 59, 60 and 61 were identified by a restrictionenzyme cleavage map and sequence analysis. The plasmids were designated“pCR-AAT7800lcr,” “pCR-AFP3800lcr” and “pCR-E6lcr,” respectively.

Meanwhile, in order to isolate the hepatocyte control region (HCR) andminimum structure HCR(HCRm) of human apolipoprotein E (ApoE) genereported by Dang Q., PCR was performed using primer sets of SEQ ID NOS:34 and 35 and SEQ ID NOS: 34 and 36, and the resulting PCR products wereinserted into pCR2.1-TOPO vectors. The HCR and HCRm of the human ApoEgene were identified by a restriction enzyme cleavage map and sequenceanalysis. The plasmids were designated “pCR-ApoEHCR” and “pCR-ApoEHCRm,”respectively. Schematic diagrams of these plasmids are shown in FIG.11A. Further, a combination of HCRm with the enhancer and promoter ofthe AAT gene was inserted into a pBluescript II vector to constructpBS-HCRm-AATenh/pro (HmA), and a combination of HCR with the promoter ofthe AAT gene was inserted into a pBluescript II vector to constructpBS-HCR-AATpro (HA). Schematic diagrams of these plasmids are shown inFIG. 11B.

<Step 2> Analysis of Gene Expression Efficiency by LCRs

The effects of the combination of the LCRs isolated in <step 1> with apromoter, an enhancer and an intron on FIX expression efficiency wereevaluated.

In detail, the pCR-AAT108lcr, pCR-AAT7800cr, pCR-AFP3800lcr, pCR-E6lcr,pCR-HCR and pCR-HCRm plasmids prepared in <step 1> were digested withHindIII/EcoRV, and inserted into HindIII/EcoRI (blunt-ended later)restriction sites of the pBS-PF prepared in <step 2> of Example 1 toobtain pBS-AAT108-PF, pBS-AAT7800-PF, pBS-AFP3800-PF, pBS-E6-PF,pBS-HCR-PF and pBS-HCRm-PF, respectively. These plasmids were digestedagain with KpnI and NotI, and inserted into KpnI/NotI restriction sitesof the pBS-CMV-hFIXUTR-Syn1PLA prepared in <step 5> of Example 2 toobtain pBS-AAT108-PF-hFIXUTR-Syn1PLA, pBS-AAT7800-PF-hFIXUTR-Syn1PLA,pBS-AFP3800-PF-hFIXUTR-Syn1PLA, pBS-E6-PF-hFIXUTR-Syn1PLA,pBS-HCR-PF-hFIXUTR-Syn1PLA and pBS-HCRm-PF-hFIXUTR-Syn1PLA. Schematicdiagrams of these plasmids are shown in FIG. 12A.

The plasmids were digested with KpnI and SalI, and inserted intoKpnI/SalI restriction sites of pTRUF6 adeno-associated virus vectors toobtain expression vectors AAT108-PF-hFIXUTR-Syn1PLA,AAT7800-PF-hFIXUTR-Syn1PLA, AFP3800-PF-hFIXUTR-Syn1PLA,E6-PF-hFIXUTR-Syn1PLA, HCR-PF-hFIXUTR-Syn1PLA andHCRm-PF-hFIXUTR-Syn1PLA. The plasmid DNAs of the expression vectors wereinjected into mouse tail veins according to the same method as in <step3> of Example 2, and the hFIX protein expression level was measured byELISA. The results are shown in FIG. 12B.

As shown in FIG. 12B, at two days after the injection, theLCR-containing expression vectors showed up to 7-fold higher hFIXexpression level than CMV-hFIXUTR-Syn1PLA (AAT7800-PF-hFIXUTR-Syn1PLA7,640 ng/ml versus CMV-hFIXUTR-Syn1PLA 1,090 ng/ml). Particularly, theAAT108-PF-hFIXUTR-Syn1PLA continuously expressed hFIX of 390 ng/ml ormore until 4 weeks after the injection, and such a expression level wasup to 1.6-fold higher than that (250 ng/ml) of the HCR-PF-FIX-Syn1PLA.This result shows that the AAT108 intron is very suitable for sustainedexpression of hFIX.

Example 4 Evaluation of Therapeutic Efficacy of the Inventive ExpressionVector for Hemophilia B

An expression vector AAT108-PAF-hFIXUTR-Syn1PLA was constructed usingpBS-PAF prepared in <step 2> of Example 1 according to the same methodas in <step 2> of Example 3. Further, the pBS-CMV-hFIXUTR-ΔNALconstructed in <2-4> of Example 2 was digested with NotI and SalI, andinserted into NotI/SalI restriction sites of theAAT108-PF-hFIXUTR-Syn1PLA and AAT108-PAF-hFIXUTR-Syn1PLA constructed in<step 2> of Example 3 to obtain expression vectorsAAT108-PF-hFIXUTR-ΔNAL and AAT108-PAF-hFIXUTR-ΔNAL.

Meanwhile, the pCR-AAT enh/pro of <step 1> of Example 1 was digestedwith SpeI (blunt-ended later)/NotI, and inserted into the EcoRV/NotIrestriction sites of the pCR-HCRm of <step 2> of Example 3 to obtain apCR-HCRm-AATenh/pro expression vector (HmA). The pCR-HCRm-AATenh/pro(HmA) expression vector was digested with KpnI and NotI, and insertedinto KpnI and NotI restriction sites of CMV-hFIXUTR-ΔNAL constructed in<Step 5> of Example 2 to obtain a HmA-hFIXUTR-NAL expression vector.

The pCR-AAT enh/pro described in <step 1> of Example 1 was digested withBglII (blunt-ended later)/NotI, and inserted into the EcoRV/NotIrestriction sites of pCR-HCR described in <step 2> of Example 3 toobtain a pBS-HCR-AATpro (HA) expression vector. The pBS-HCR-AATpro (HA)expression vector was digested with KpnI/NotI, and inserted intoKpnI/NotI restriction sites of CMV-hFIXUTR-1.4kbFIXint described in<step 3> of Example 2 to obtain a HA-hFIXUTR-1.4kbFIXint expressionvector.

Schematic diagrams of the expression vectors are shown in FIG. 13A.

25 μg of each plasmid DNA of the AAT108-PF-hFIXUTR-ΔNAL,AAT108-PAF-hFIXUTR-ΔNAL, HmA-hFIXUTR-ΔNAL, and CMV-hFIXUTR-ΔNAL(prepared in <step 5> of Example 2) expression vectors was injected intoa tail vein of hemophilia B mouse, and the hFIX protein concentrationand the blood clotting activity were measured by ELISA.

As shown in FIG. 13B, the hFIX expression level induced by theAAT108-PAF-hFIXUTR-ΔNAL expression vector was 9.8% at day 1 after theinjection, 30.6% at day 2, 82% at day 7, 108% at week 2 and 208% at week9, relative to that induced by the HmA-hFIXUTR-ΔNAL expression vector.Further, the hFIX expression level induced by the AAT108-PF-hFIXUTR-ΔNALexpression vector was 9.0% at day 1 after the injection, 19.2% at day 2,41.9% at day 7, 56.9% at week 2 and 73.0% at week 9, relative to thatinduced by the HmA-hFIXUTR-ΔNAL expression vector.

The blood clotting activities of normal mouse and hemophilia B miceinjected with hFIX expression vectors were measured by an activatedpartial thromboplastin time (APTT) method as follows. FIX-deficientplasma, 10-fold diluted mouse-derived plasma and activated actin (each50 μl) were mixed and incubated at 37° C. for three minutes. CaCl₂ wasadded thereto, and the time taken for sample clotting was measured witha blood clotting detector KC10A (Amelung). The blood clotting time ofthe normal mouse was about 44 seconds, and that of the hemophilia Bmouse was about 65 seconds. As shown in FIG. 13C, the hemophilia B miceadministrated with the hFIX expression vectors showed about 80% of thenormal blood clotting time until 3 days after the injection. After 7days, the blood clotting time of the CMV-hFIXUTR-ΔNAL-administrated micewas returned to the level before the injection, but the miceadministrated with the AAT108-PF-hFIXUTR-ΔNAL andAAT108-PAF-hFIXUTR-ΔNAL exhibited an improved blood clotting time of 50to 60 seconds.

Further, the clotting activity of the samples was calculated accordingto a standard curve of standard activity versus standard clotting time.As shown in FIG. 13D, the blood clotting activities of the hemophilia Bmice treated with the AAT108-PF-hFIXUTR-ΔNAL and AAT108-PAF-hFIXUTR-ΔNALexpression vectors were respectively 34.1% and 29.3% at week 8 after theinjection, relative to that of normal mice. Particularly, the bloodclotting activity of the mice treated with the AAT108-PAF-hFIXUTR-ΔNALexpression vector was 31.9% at week 8 after injection, relative to thatof the normal mice.

Meanwhile, 2×10⁹ IP of recombinant adeno-associated viruses using theHA-hFIXUTR-1.4kbFIXint, AAT108-PF-hFIXUTR-ΔNAL, AAT108-PAF-hFIXUTR-ΔNAL,and CMV-hFIXUTR-1.4kbFIXint expression vectors according to the samemethod as in <step 3> of Example 2 were injected into portal veins ofhemophilia B mice, and the hFIX protein concentration and the bloodclotting activity were measured by ELISA and APTT method as describedabove. The results are shown in FIGS. 14A and 14B.

As shown in FIG. 14A, the AAT108-PF-hFIXUTR-ΔNAL-carrying virusesinduced hFIX protein of up to 2,379 ng/ml, and theAAT108-PAF-hFIXUTR-ΔNAL-carrying viruses induced hFIX protein of up to1,431 ng/ml. On the other hand, the hFIX expression levels induced bythe control viruses, i.e., the CMV-hFIXUTR-1.4kbFIXint-carrying virusesand the HA-hFIXUTR-1.4kbFIXint-carrying viruses were up to 1,542 ng/mland 380 ng/ml, respectively. Particularly, theAAT108-PF-hFIXUTR-ΔNAL-containing viruses induced hFIX expression of 500ng/ml or more until 68 weeks after the injection. This result shows thatLCR-containing expression vectors according to the present invention aresuitable for gene therapy requiring long-term gene expression.

As shown in FIG. 14B, the mice administrated with rAAV carryingAAT108-PF-hFIXUTR-ΔNAL exhibited 59.1% or more of clotting activity ofnormal mice until 7 weeks after the injection.

The above results show that the inventive gene expression systems areeffective for stable and sustained expression required for the treatmentof hemophilia.

Example 5 Construction of Liver-Specific Expression Vectors IncludingKringle Domain KIV9-KIV10-KV(LK68) Gene of Anti-Angiogenesis Protein Apo(a) and Evaluation of LK68 Protein Expression Efficiency

In order to introduce ΔNAL intron and FIX UTR into a LK68 expressionvector, first, PCR was performed using pAAV-LK68 (Patent No.KR10-0681762) as a template, primer sets of SEQ ID NOS: 37 and 38 andSEQ ID NOS: 39 and 40, and Ex-Taq to obtain a FIX 5′UTR-signal sequencefragment and an LK68-FIX 3′UTR fragment. The PCR condition was asfollows: initial denaturation at 94° C. for five minutes; 30 cycles ofdenaturation at 94° C. for one minute, annealing at 60° C. for oneminute and extension at 72° C. for one minute; and final extension at72° C. for three minutes. The 5′UTR-signal sequence fragment and theLK68-3′UTR fragment were respectively inserted into NotI/XhoI andAatII/SalI restriction sites of the pBS-hFIXUTR-ΔNAL vector prepared in<2-4> of Example 2. The desired DNA fragments were identified by arestriction enzyme cleavage map and sequence analysis, and the plasmidwas designated “pBS-LK68-ΔNAL-FUTR.” The LK68-ΔNAL-FUTR fragmentobtained by treating the pBS-LK68-ΔNAL-FUTR plasmid with restrictionenzymes NotI and SalI was inserted into NotI/SalI restriction sites ofthe AAT108-PF-hFIXUTR-NAL prepared in Example 4 to obtain an expressionvector AAT108-PF-LK68-ΔNAL-FUTR. The above procedure was repeated in thepresence of different LCRs or in the absence of LCR to obtain expressionvectors E6-PF-LK68-ΔNAL-FUTR, AAT7800-PF-LK68-ΔNAL-FUTR,AFP3800-PF-LK68-ΔNAL-FUTR, HCRm-PF-LK68-ΔNAL-FUTR, HCR-PF-LK68-ΔNAL-FUTRand PF-LK68-ΔNAL-FUTR. The cleavage maps of the expression vectors areshown in FIG. 15A.

The expression vectors were inserted into mouse tail veins according tothe same method as in <step 3> of Example 2. Blood samples werecollected from the mice, and the expression level of LK68 protein wasmeasured by ELISA.

As shown in FIG. 16A, among LCRs, AAT108 showed the highest geneexpression efficiency. Particularly, the LK68 expression efficiency ofthe AAT108-PF-LK68-ΔNAL-FUTR expression vector was 158.4% at day 1 afterthe injection, 104.8% at day 2, 155.8% at day 7, 160.2% at week 2,162.7% at week 3 and 144.1% at week 4, relative to that of thePF-LK68-ΔNAL-FUTR expression vector.

Further, the above procedure was repeated to obtain expression vectorsincluding expression cassettes AAT108-PF-LK68, AAT108-PF-LK68-FUTR,AAT108-PF-LK68-ΔNAL-FUTR, AAT108-PAF-LK68, AAT108-PAF-LK68-FUTR andAAT108-PAF-LK68-ΔNAL-FUTR. Schematic diagrams of the expression vectorsare shown in FIG. 15B.

The expression vectors were injected into mouse tail veins according tothe same method as in <step 3> of Example 2. Blood samples werecollected from the mice, and the expression level of LK68 protein wasmeasured by ELISA.

As shown in FIG. 16B, the LK68 expression efficiencies of theAAT108-PF-LK68-FUTR and AAT108-PF-LK68-ΔNAL-FUTR expression vectors wererespectively 197.7% and 425.4% at day 1 after the injection, 478.6% and891.2% at day 2, 233.2% and 319.3% at day 7, 203.0% and 253.7% at week2, 113.8% and 243.3% at week 3 and 170.4% and 277.6% at week 4, relativeto that of the AAT108-PF-LK68 expression vector. The LK68 expressionefficiencies of the AAT108-PAF-LK68-FUTR and AAT108-PAF-LK68-ΔNAL-FUTRexpression vectors were respectively 147.0% and 344.6% at day 1 afterthe injection, 351.0% and 824.5% at day 2, 220.2% and 260.3% at day 7,140.8% and 203.0% at week 2, 146.5% and 288.4% at week 3 and 177.8% and323.8% at week 4, relative to that of the AAT108-PAF-LK68 expressionvector.

These results show that gene expression in the presence of intron and/orUTR is sustained at a high level, as compared to that in the absence ofintron and UTR.

Example 6 Evaluation of Expression Efficiency of Kringle DomainKIV9-KIV10-KV (LK68) of Anti-Angiogenesis Protein Apo (a) by UTR andIntron in Cells

LK68 and LK68-ΔNAL-FUTR fragments obtained by digesting theAAT108-PF-LK68 and AAT108-PF-LK68-ΔNAL-FUTR vectors described in Example5 with NotI and SalI were inserted into adenovirus shuttle vectorspENTR2B (Invitrogen, Carlsbad, Calif.) to obtain pENTR-LK68 andpENTR-LK68-ΔNAL-FUTR vectors. These shuttle vectors and a target vectorpAd/CMV/V5-DEST (Invitrogen, Carlsbad, Calif.) were subjected to invitro homologous recombination with Clonase™ (Invitrogen, Carlsbad,Calif.) to obtain pAd-CMV-LK68 and pAd-CMV-LK68-ΔNAL-FUTR vectors. ThepAd-CMV-LK68 and pAd-CMV-LK68-ΔNAL-FUTR vectors were linearized with aPacI restriction enzyme, and transfected into a HEK 293 kidney cell lineto obtain replication-defective adenoviruses rAd-LK68 and rAd-LK68_UNcarrying CMV-LK68 and CMV-LK68-ΔNAL-FUTR, respectively. The rAd-LK68 andrAd-LK68_UN were infected into HEK 293 kidney cell line, and theexpression level of LK68 protein was measured.

The infection into the HEK 293 kidney cell lines was performed atdifferent multiplicity of infection (MOI) of 0.1, 0.5, 2 and 10. Thecells and media were collected at 2 days after adenovirus infection. Theexpression level of LK68 protein was measured by electrophoresis usinganti-LK68 antibody. The results are shown in FIGS. 17A and 17B.

As shown in FIGS. 17A and 17B, at MOI 0.1, 0.5 and 2, the expressionlevels of LK68 protein were low and equal, however at MOI 10, theexpression level of LK68 protein induced by rAd-LK68_UN was increased420% in cells and 242% in media, as compared with that induced byrAd-LK68.

These results show that gene expression efficiency in cells and media ishigher in the presence of UTR and intron than in the absence of UTR andintron.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made and also fall within the scope of the inventionas defined by the claims that follow.

1. An isolated polynucleotide having the nucleotide sequence as setforth in SEQ ID NO:
 49. 2. An expression cassette comprising: apromoter; a coding sequence operably linked to and under control of thepromoter; and an intron, operably linked to the coding sequence, havingthe nucleotide sequence as set forth in SEQ ID NO:
 49. 3. The expressioncassette of claim 2, wherein the promoter is selected from the groupconsisting of polynucleotides having nucleotide sequences as set forthin SEQ ID NOS: 41, 42 and
 43. 4. The expression cassette of claim 2,further comprising at least one of enhancers, operably linked to thecoding sequence, having nucleotide sequences as set forth in SEQ ID NOS:44 and
 45. 5. The expression cassette of claim 2, further comprising atleast one of local control regions, operably linked to the codingsequence, having nucleotide sequences as set forth in SEQ ID NOS: 58 to61.
 6. The expression cassette of claim 2, further comprisinguntranslated regions, operably linked to the coding sequence, havingnucleotide sequences of SEQ ID NOS: 62 and 63 at 5′ and 3′-ends of thecoding sequence, respectively.
 7. The expression cassette of claim 2,wherein the coding sequence is a nucleotide sequence encoding aliver-specific protein selected from the group consisting of albumin,α-fetoprotein, α-glucosidase, α1-antitrypsin, antithrombin,lipoproteins, ceruloplasmin, coagulation factor VII, coagulation factorVIII, coagulation factor IX, erythropoietin, fibrinogen,glucocerebrosidase, haptoglobin, IGF-1, insulin, plasminogen,prothrombin and transferrin.
 8. An expression vector comprising theexpression cassette of claim
 2. 9. The expression cassette of claim 4,further comprising at least one of local control regions, operablylinked to the coding sequence, having nucleotide sequences as set forthin SEQ ID NOS: 58 to
 61. 10. The expression cassette of claim 9, furthercomprising untranslated regions, operably linked to the coding sequence,having nucleotide sequences of SEQ ID NOS: 62 and 63 at 5′ and 3′-endsof the coding sequence, respectively.
 11. The expression cassette ofclaim 4, further comprising untranslated regions, operably linked to thecoding sequence, having nucleotide sequences of SEQ ID NOS: 62 and 63 at5′ and 3′-ends of the coding sequence, respectively.
 12. The expressioncassette of claim 5, further comprising untranslated regions, operablylinked to the coding sequence, having nucleotide sequences of SEQ IDNOS: 62 and 63 at 5′ and 3′-ends of the coding sequence, respectively.13. An expression vector comprising the expression cassette of claim 4.14. An expression vector comprising the expression cassette of claim 5.15. An expression vector comprising the expression cassette of claim 6.16. An expression vector comprising the expression cassette of claim 9.17. An expression vector comprising the expression cassette of claim 10.18. An expression vector comprising the expression cassette of claim 11.19. An expression vector comprising the expression cassette of claim 12.